BioMEMs technologies derive from novel developments in materials and micro/nanofabrication methods. The prior art cited teaches how to use these integrated technologies (e.g., microfluidics, electronics and photonics, and biocompatible surface chemistries) to create fluidic systems that are well-suited to carry, manipulate, detect, analyze and process biological molecules, organelles, and whole cells. These systems also benefit from new light sources derived from semiconductors and solid state devices to enable efficient new tools for bioanalysis because they are small, easily integrated with microfluidics, and are well-adapted to microscopy and spectroscopy for imaging, flow spectrocytometry, and high speed analysis.
This invention teaches methods for analyzing data from resonant optical devices used to measure molecules, organelles, and cells (collectively called bioparticles here). Measurements of any optical property of a bioparticle from other device also benefits from this analysis. The an optical property called Δλ that represents the difference between a measured resonance wavelength of a bioparticle in an optical cavity and a reference wavelength. The discussion can be generalized to other optical measurements from a biosensor.
The invention uses optical resonators to measure biophysical properties of bioparticles. The optical resonator can take many different forms by using different materials, geometries, wavelength regions, surface treatments or means for coupling light into or out of the resonator.
Generally, the resonator is a reflective structure comprising a cavity space for fluidic specimens. It comprises a means for internal or external light generation and a means for coupling light into and out of the structure. It allows the establishment of a resonance of light waves within the cavity and means to measure the resonance condition in the absence or presence of a specimen. The resonator may be constructed with materials such as dielectrics, metals, glasses, plastics, semiconductors, polymers or the like. The structure may take on different geometrical forms such as planar, box-like, rod, cylindrical, ring, spherical, or more complex shapes. It includes structures like waveguides, photonic lattices, periodic bandgap materials, or holey fibers. The geometry may include nanostructured components like quantum dots, arrays, wires, or layers. It may comprise surface treatments such as coatings, chemically functionalized surfaces, layers, processing, or thermal treatments to enhance the cavity optical performance or facilitate fluidic transport of specimens into and out of the cavity. In the discussion to follow, a planar mirror cavity forming a laser is used to illustrate the operation and method of analyzing bioparticles. However, the invention is not limited to a laser or limited by these choices of material and geometry.
In prior art, a biocavity laser device was developed for the analysis of cells. It relies on recent semiconductor micro/nanotechnology that has reduced the size of a laser to ultra small dimensions (tens of nanometers to microns) that match the size of bioparticles. The laser was integrated with a microfluidic chip to flow and analyze populations of bioparticles. It has shown the potential to probe the human immune system, characterize genetic disorders, and distinguish cancerous from normal cells. Most importantly, cells can be analyzed immediately after they are removed from the body. There are no time delays or difficulties associated with chemical fixing or tagging cells with stains or fluorescent markers. The applications of such a portable biological sensing device are potentially far-reaching, including reatime biopsy to enable surgeons and their patient's confidence that all of the diseased tissue had been removed during surgery.
The laser has recently been extended to the analysis of small bioparticles (smaller than the wavelength of light) such as organelles using a phenomenon called “nano-squeezed light.” The laser light is “nano-squeezed” through the organelle and a single lasing mode is supported. This results in a discrete band of laser light with a simpler spectrum to analyze compared to whole cells.
The laser works on the principle that the speed of light through a biological cell is slowed by the presence of biomolecules. By flowing a fluid, cells, or bioparticles through a semiconductor microcavity laser, these decreases in light speed can be registered as small wavelength red-shifts in the emitted laser output spectrum. The biocavity laser is used to measure this biophysical optic parameter Δλ, a laser wavelength shift relating to the optical density of cell or organelles that reflects its size and biomolecular composition. As such, Δλ is a powerful parameter that rapidly interrogates the biomolecular state of single cells and organelles. The laser shift Δλ can be viewed as a wavelength detuning (or alternately as a frequency detuning Δω) of the cavity resonance in dimensionless units as δ=Δλ/λ=Δω/ω where λ and ω are the fluid-filled cavity (without cell) resonance wavelength and frequency, respectively. Experimentally, Δλ is measured in nanometers as the difference between a longitudinal laser mode of the fluid-filled cavity and the red-shifted laser wavelength produced by flowing cells or organelles (e.g. mitochondria) through the cavity.
Because of its importance, it is essential to properly interpret the measurement and make highly accurate measurements of Δλ. This invention improves upon the interpretation and accuracy of measuring Δλ in the following ways:
1. It provides a means for interpreting the measurement. The invention solves a technical difficulty in knowing exactly how the wavelength shift Δλ relates to the biophysical properties (i.e. the diameter and refractive index) of the particle. Basically, the problem centers on determining the resonance frequencies of a particle in a planar active cavity with optical gain. The problem is not exactly solvable, but recent experiments on a variety of particles in various cavities have shown that a good approximation is given by a simple empirical relationshipΔλ=kdΔn  (1)where Δn is the difference in refractive index between the particle and its surrounding fluid, d is the average particle diameter, and k is a constant relating to the geometry of the cavity. These results are an improvement over prior art that lead to inaccuracies in determining biophysical parameters.2. It provides a means for predicting the distribution of Δλ among a population. Using the empirical relationship in Eq. 1, it is possible to develop statistical methods to predict how the probability distribution of Δλ should depend on the biophysical quantities d and Δn. Experiments show that the probability distribution of diameters for a given type of bioparticle is very often approximated by a normal distribution function. This is not necessarily the case for the probability distribution of Δn.3. It provides a means for improving the accuracy of the measurement of Δλ. The invention solves another technical difficulty relating to errors in the measurement of Δλ. Δλ depends on the accurate measure of both the lasing mode wavelength the reference wavelength. Because of possible instrumental drift effects, the bioparticle resonance wavelength and/or reference wavelength may change with time and cause errors in the computation of Δλ. It is important to provide a means to correct for this drift to allow accurate computation of Δλ.4. It provides a means for absolute calibration of the measurement of Δλ. The measurement of the Δλ can sometimes be complicated by the calibration of the zero of measurement. Knowing the relationship between the measured property Δλ and the biophysical properties can help determine the zero calibration of the measurement. This invention provides a method for calibrating the measurement of Δλ.5. It provides a new, simpler apparatus for measuring optical properties of bioparticles. The invention provides an advantage over prior art to extract both size and refractive index properties of the bioparticle using multiple measurements in a single apparatus.
6. It provides a new method of operation for very small bioparticles of size less than the wavelength of light. The invention provides a new means for measuring physical properties of very small particles using light fluctuations arising from the interaction of bioparticles with the resonant light waves within the cavity.
7. It provides a new method for detecting, manipulating and separating bioparticles.
The invention makes use of arrays of micro- or nanocavity resonators acting as lasers to probe living cells and bioparticles. The arrays can also act as optical traps to simultaneously trap and analyze bioparticles or separate them from other species.